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Plasmid DNA Sent into Space
Overview On earth, there are a multitude of organisms that can survive at extreme conditions. Environments with extreme heat, radiation exposure, intense pressure and acidity can all host biologically active organisms. In space, the conditions can be far more extreme, and the ability of basic molecular building blocks and biomarkers to remain stable and functional in these conditions is the basis for life. It can be assumed that there is life on other planets, and the potential movement of these microorganisms from one planet to another via meteorites could provide insights into the spread of and origin of life itself. A meteorite entering a planets atmosphere undergoes extreme conditions for a matter of seconds, however the interior of that meteorite is highly insulated, and it has been determined that the interior of some martian meteorites remains below 100 C which many spores produced on earth would be able to survive. Sounding rockets, which are sent into suborbital space provide an appropriate platform for testing the stability of various biomolecules as they leave and re-enter earth's atmosphere. Methods In 2011, artificial plasmid DNA carrying a fluorescent marker and a kanamycin resistance cassette was attached to different positions on a sounding rocket, that was propelled into subor bital space. The plasmid, expression vector pEGFP-C3, is 4727bp, was transformed in E. coli, and grown on an LB medium with kanamycin. 50-100 micrograms of plasmid in a Tris buffer was attached to 15 different location on the structure. 4 were applied directly to the surface, 8 were applied into the grooves of the screws of the payload, and 3 samples were applied to different locations on the bottom side. This mission, TEXUS-49, was launched nine days after the plasmid DNA was applied, on March 29th 2011, from the European Space and Sounding Rocket Range in Sweden. The flight reached an altitude of 268 km, 378 s of microgravity, and a peak thrust acceleration of 17.6g during reentry. Temperature reached two peaks at 118 and 130 C on the inside of the recovery module and outer gas temperatures of over 1000 C were estimated at the application sites. Assessment of DNA Recovery and Quantitative Values The DNA was recovered by dissolving the buffer, and the concentration and purity was determined using spectrophotometry. Of the fifteen experimental sample, twelve showed a high degree of purity with 260/280 nm ratios between 1.71 and 1.85. The other three samples showed lower purity and protein contamination with ratios between 1.56 and 1.68, this protein contamination likely occurred during landing and travel throughout the atmosphere. The recovery efficiency ranged between 4.9% and 53.4% for the samples, and on average the bottom side of the payload was where the highest amount of DNA was recovered. Functionality After testing the quantity and quality of the recovered DNA the functional activity of the plasmid DNA was tested by measuring the colonies formed per nanogram of transformed DNA after the plasmid was transformed back into E. coli cells. Only one of the surface area samples retained any integrity at all at 4%, while all of the screw heads and bottom side samples retained some integrity as being biologically active. One of the bottom side samples retained 33% of its integrity which is significant considering the positive control was 48%. a second test of functionality was performed by transfecting mouse fibroblasts with plasmid DNA from each of the three different application sites. One day after transfection the samples were analyzed microscopically for the expression of the fluorescent marker on the plasmid. For all transfected DNA samples there was similar GFP expression indicating that the circular plasmid stayed intact post-flight. References Thiel CS, Tauber S, Schütte A, Schmitz B, Nuesse H, et al. (2014) Functional Activity of Plasmid DNA after Entry into the Atmosphere of Earth Investigated by a New Biomarker Stability Assay for Ballistic Spaceflight Experiments. PLoS ONE 9(11): e112979. doi:10.1371/journal.pone.0112979